The basic structural unit of an immunoglobulin features two light chains and two heavy chains. Each light chain is made up of a variable region and a constant region and is associated with a corresponding heavy chain. Each heavy chain is composed of a variable region and three constant regions, where the constant regions of the heavy chain are collectively longer than the constant region of the light chain and extend through a hinge region.
The Fab fragment of an immunoglobulin includes the entire light chain of the immunoglobulin and the variable region and a corresponding length of the constant region of the heavy chain. The lengths of both heavy chains extending beyond the Fab fragment and hinge region constitute the Fc fragment of the immunoglobulin. The F(ab').sub.2 fragment of an immunoglobulin includes both light chains and a length of both heavy chains, extending through the hinge region thereof and joined by disulfide bonds. Immunoglobulin fragments are useful in a number of therapeutic and diagnostic applications and exhibit greater utility than whole immunoglobulin for some of those purposes.
Fab and F(ab').sub.2 fragments are formed by proteolytic fragmentation reactions. Exemplary conventional fragmentation reactions are partial digestion with the proteolytic enzymes papain and pepsin. Papain treatment theoretically yields Fab fragment. Under some circumstances for some immunoglobulins, however, papain treatment results in low yields of Fab fragment. One method to increase the yield of Fab fragment is to conduct the fragmentation in the presence of cysteine. While the yield of Fab may be increased by this method, the quality of the Fab fragment produced may diminish, i.e., the Fab fragment produced may not be homogeneous. Pepsin treatment is used to produce F(ab').sub.2 fragment; however, problems exist in production of this fragment for certain immunoglobulins, where pepsin treatment results in the production of one Fab fragment and one Fab/Fc fragment.
Monoclonal antibodies are immunoglobulins as well as glycoproteins, i.e., proteins having sugar moieties covalently bound thereto through N and/or O glycosyl bonds. Carbohydrate moieties are generally bound to immunoglobulins at a location or locations in the Fc region. Carbohydrates may also be found at the hinge region or on the Fab portions of an immunoglobulin, however. Carbohydrate moieties located on immunoglobulins can contribute to the biological activity of the immunoglobulins in a number of ways, including the mediation of intracellular and intercellular immunoglobulin recognition.
The oligosaccharide units of glycoproteins may be varied and complex. The properties of these oligosaccharide units depend upon the composition of the sugar residues contained therein, including the anomeric configuration of each residue, the sequence of the sugar residues, the pattern of glycosidic linkages within the sequence, and the nature of the linkage of the oligosaccharide units to the protein.
Oligosaccharide moieties located on immunoglobulins are often capped with a terminal sialic acid moiety. As discussed in PCT Application No. US86/00495, published on Sept. 11, 1987, such immunoglobulins are not rapidly cleared from the bloodstream, because there appears to be no sialic acid specific receptor located on cells responsible for bloodstream clearance. In this PCT application, sialic acid residues are attached to a protein to be administered to a patient, thereby increasing the in vivo halflife and increasing the stability of that protein.
Desialylation reactions are used in U.S. Pat. No. 4,859,449 to expose underlying sugar residues in efforts to control the halflife of administered protein. Like the PCT Application described above, the methods, conjugates and kits of this patent are constructed in accordance with the notion that glycoproteins having exposed sugar residues recognized by certain receptors on cells active in blood clearance are cleared from the bloodstream more rapidly than glycoproteins having oligosaccharide units capped with sialic acid.
Although heavy chain Ig polypeptides encoded by a single constant region heavy chain gene typically migrate as a single molecular weight band ranging from about 52,000 to about 56,000 daltons under reduced conditions on SDS-polyacrylamide gel electrophoresis, double and triple heavy chain bands have been observed for murine immunoglobulins of the IgG.sub.2b and IgG.sub.2a isotypes (Kohler et al., 1978, European J. Immunology, 8: 82-88 and Leatherbarrow, R. J. et al., 1985, Molecular Immunology 22; 4, pp. 407-415). This observed heterogeneity in molecular weight of the heavy chains in these immunoglobulin isotypes has been attributed to differing glycosylation of the respective heavy chains.